Food Chemistry 138 (2013) 950–955

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Food Chemistry

journal homepage: www.elsevier .com/locate / foodchem

Protective effect of marine algae phlorotannins against AAPH-induced oxidativestress in zebrafish embryo

Min-Cheol Kang a,1, Seon Heui Cha b,1, W.A.J.P. Wijesinghe a, Sung-Myung Kang a, Seung-Hong Lee c,Eun-A. Kim a, Choon Bok Song a, You-Jin Jeon a,d,⇑a Department of Marine Life Sciences, Jeju National University, Jeju 690-756, Republic of Koreab School of Biology and Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technolgy, GA 30332, United Statesc School of Medicine, Jeju National University, Jeju 690-756, Republic of Koread Marine and Environmental Research Institute, Jeju National University, Jeju 695-814, Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 August 2012Received in revised form 29 October 2012Accepted 2 November 2012Available online 12 November 2012

Keywords:ZebrafishIn vivo modelMarine polyphenolOxidative stressPhlorotannins

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.11.005

⇑ Corresponding author at: Department of MarineUniversity, Jeju 690-756, Republic of Korea. Tel.: +823493.

E-mail address: Youjinj@jejunu.ac.kr (Y.-J. Jeon).1 These authors equally contributed to this work.

In this study the protective effect of phlorotannins, including phloroglucinol, eckol, dieckol, eckstolonoland triphloroethol A, isolated from brown algae Ecklonia cava was investigated against AAPH-inducedoxidative stress toxicity in zebrafish embryos. Zebrafish embryos were exposed to AAPH and comparedwith other groups that were co-exposed with phlorotannins until 2-days post-fertilisation. All phlorotan-nins scavenged intracellular ROS and prevented lipid peroxidation and reduced AAPH-induced cell deathin zebrafish embryos. Negative changes in morphological phenomena, such as pericardial oedema, yolksac oedema, and growth retardation in zebrafish embryos exposed to AAPH were not observed in groupsexposed to phlorotannins. These results clearly indicate that phlorotannins possess prominent antioxi-dant activity against AAPH-mediated toxicity and might be potential therapeutic agents for treating orpreventing several diseases implicated with oxidative stress. This study provides a useful tool for exam-ining the protective effect of antioxidants against AAPH-induced oxidative stress in an alternative in vivomodel.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Biological properties of natural products have attracted theattention of scientists for a long time and in vivo studies of partic-ular biological activities have been demonstrated in various animalmodels (Kang et al., 2012). A model organism should carrytechnical and practical advantages for studying specific biologicalprocesses, effects and mechanisms. Recently, numerous advanta-ges of the zebrafish model system have been shown in in vivostudies and zebrafish (Danio rerio) has emerged as a highly usefulvertebrate model organism (Kimmel, Ballard, Kimmel, Ullmann, &Schilling, 1995). Development in zebrafish is very similar to theembryogenesis in higher vertebrates, including humans. Zebrafishare well suitable for functional genomics studies (Driever et al.,1996). For a vertebrate model system, zebrafish offer the uniquecharacteristics of being simple to maintain and also viable for theassessment of drugs and/or small molecules as well as cell studies(Choi et al., 2007).

ll rights reserved.

Life Sciences, Jeju National64 754 3475; fax: +82 64 756

Human diseases resulting from oxidative stress can be causedby free radicals and natural antioxidants can act as free radicalscavengers (Chang, Wu, & Chiang, 2007). Free radical-mediatedoxidative stress and antioxidants are widely discussed in manycurrent research areas (Valko et al., 2007). Free radicals can causedamage of tissue by reacting with other chemicals in the body.Therefore, looking for functional materials that possess antioxidantactivity has become a hot research subject (Lindmark-Mansson &Akesson, 2000).

Marine organisms have been proven to be a rich source of struc-turally novel and biologically active secondary metabolites (Heo etal., 2008). Marine algae have many phytochemicals with variousbioactivities including antioxidant, anti-inflammatory and anti-cancer (Lordan, Ross, & Stanton, 2011). Marine algae are alsoknown to be rich in vitamins, minerals, polysaccharides, proteins,and polyphenols (Kuda, Tsunekawa, Goto, & Araki, 2005).

Ecklonia cava (class, Phaeophyceae; family, Lessoniaceae; order,Laminariales, E. cava) is a brown alga which grows abundantly inthe sub-tidal regions of Jeju Island, Korea. E. cava harbours a richersupply of total phenolic compounds, including phlorotannins, thanother brown seaweeds (Heo, Park, Lee, & Jeon, 2005; Kim et al.,2006). These phenolic secondary metabolites, such as phloroglu-cinol (1,3,5-trihydroxybenzene), eckol (a trimer of phloroglucinol),

M.-C. Kang et al. / Food Chemistry 138 (2013) 950–955 951

6,6-bieckol (a hexamer), dieckol (a hexamer), phlorofucofuroeckol(a pentamer) and triphlorethol-A, have been associated with a vari-ety of biological activities (Kang et al., 2006). Recently, an expand-ing body of evidence revealed that E. cava has radical scavenging,matrix metalloproteinase inhibitory, antioxidative, anti-inflamma-tory, immunomodulatory, HIV-1 reverse transcriptase, and anti-asthmatic activities (Ahn et al., 2008; Jung et al., 2009).

Nevertheless, relatively little research regarding the in vivo effi-cacy of E. cava phlorotannins has been conducted thus far. Thisstudy evaluated the potential protective efficacy of five phlorotan-nins, dieckol (DK), eckstolonol (ES), eckol (EK), triphloroethol A(TA), and phloroglucinol (PG), on AAPH-induced oxidative stressin zebrafish embryo as an alternative in vivo model.

2. Materials and methods

2.1. Materials

The marine brown alga E. cava was collected along the coast ofJeju Island, Korea, between October 2007 and March 2008. Thesamples were washed three times with tap water to remove salt,epiphytes, and sand attached to the surface. They were then care-fully rinsed with fresh water, and maintained in a medical refriger-ator at �20 �C. Thereafter, the frozen samples were lyophilised andhomogenised using a grinder prior to extraction.

2.2. Isolation of phlorotannins from E. cava

The phlorotannins were isolated as previously described by Ahnet al. (2007) with slight modifications. Briefly, the dried E. cavapowder (500 g) was extracted three times with 80% MeOH andthen filtered. The filtrate was evaporated at 40 �C to obtain themethanol extract. The extract was then suspended in distilledwater and partitioned with ethyl acetate. The ethyl acetate fractionwas mixed with celite. The mixed celite was dried and packed intoa glass column, and eluted sequentially with hexane, methylenechloride, diethyl ether, and methanol. The diethyl ether fractionwas further purified by Sephadex LH-20 column chromatographyusing a stepwise gradient chloroform/methanol (2/1 ? 0/1) sol-vents system. The phloroglucinol, eckol, triphloroethol A, eckstolo-nol and dieckol were purified by high-performance liquidchromatography (HPLC) using a Waters HPLC system (Waters Cor-poration, Milford, MA) equipped with a Waters 996 photodiode ar-ray detector and C18 column (J’sphere ODS-H80, 150 � 20 mm,4 lm; YMC, Kyoto, Japan) by stepwise elution with methanol-water gradient (UV range: 230 nm, flow rate: 0.8 ml/min). Finally,the purified compounds were identified by comparing their 1H and13C NMR data with the literature. The chemical structures of thephlorotannins are indicated in Fig. 1.

2.3. Origin and maintenance of zebrafish

Adult zebrafish were obtained from a commercial dealer (SeoulAquarium, Korea) and 10 fish were kept in a 3-L acrylic tank underthe following conditions: 28.5 �C, with a 14/10 h light/dark cycle.Fish were fed three times a day, 6 days a week, with Tetramin flakefood supplemented with live brine shrimps (Artemia salina). Em-bryos were obtained from natural spawning that was induced inthe morning by turning on the light. Collection of embryos wascompleted within 30 min.

2.4. Waterborne exposure of embryos to phlorotannins and AAPH

From approximately 3 hour post-fertilisation (3 hpf), embryos(group = 25 embryos) were transferred to individual wells of a

24-well plate and maintained in embryo medium containing1 ml of vehicle (0.1% DMSO) or 50 lM phlorotannins for 1 h. Thenembryos were treated with 25 mM AAPH (2,20-azobis-2-methyl-propanimidamide dihydrochloride) or co-treated with AAPH andphlorotannins for up to 120 hours post-fertilisation (120 hpf).

2.5. Measurement of heartbeat rate

The heartbeat rate of both atrium and ventricle were measuredat 35 hpf to determine the sample toxicity (Choi et al., 2007).Counting and recording of atrial and ventricular contractions wereperformed for 3 min under a microscope, and results were pre-sented as the average heartbeat rate per min.

2.6. Estimation of intracellular ROS generation and image analysis

Generation of ROS in zebrafish embryos was analysed using anoxidation-sensitive fluorescent probe dye, 2,7-dichlorodihydroflu-orescein diacetate (DCFH-DA). DCFH-DA was deacetylated intra-cellularly by nonspecific esterase, which was further oxidised tothe highly fluorescent compound dichlorofluorescein (DCF) in thepresence of cellular peroxides (Rosenkranz et al., 1992). At 3–4 hpf, the embryos were treated with 50 lM phlorotannins and,1 h later, 25 mM AAPH was added to the plate. After treating with25 mM AAPH for 6 h, the embryo medium was changed and theembryos developed up to 2 dpf. The embryos were transferred into96-well plates and treated with DCFH-DA solution (20 lg/ml), andthe plates were incubated for 1 h in the dark at 28.5 �C. After incu-bation, the embryos were rinsed in fresh embryo medium andanaesthetised before visualisation. Individual embryo fluorescenceintensity was quantified using a spectrofluorometer (Perkin–ElmerLS-5B, PerkinElmer Life And Analytical Sciences, Inc., Waltham,MA) and the images of stained embryos were observed using afluorescence microscope, which was equipped with a CoolSNAP-Pro colour digital camera (Olympus, Tokyo, Japan).

2.7. Lipid peroxidation inhibitory activity and image analysis

Lipid peroxidation was measured to assess membrane damageaccording to a method described by Wang, Gong, and Zhou(2008). Morphological evaluation of the embryos was performedwith diphenyl-1-pyrenylphosphine (DPPP; Dojindo Laboratories,Kumamoto, Japan) which is a fluorescent probe for detection of cellmembrane lipid peroxidation. DPPP is non-fluorescent, but it be-comes fluorescent when oxidised. At 3–4 hpf, the embryos weretreated with 50 lM phlorotannins and, 1 h later, 25 mM AAPHwas added to the plate. After treating embryos with 25 mM AAPHfor 6 h, the embryo medium was changed and the embryos devel-oped up to 2 dpf. The embryos were transferred into 96-well platesand treated with DPPP solution (25 lg/ml), and the plates wereincubated for 1 h in the dark at 28.5 �C. After incubation, theembryos were rinsed in embryo medium and anaesthetisedbefore visualisation. Individual embryo fluorescence intensitywas quantified using a spectrofluorometer (Beckman DTX 800,Beckman Coulter, Inc., Fullerton, CA) and image of embryos wereobserved using a fluorescence microscope, which was equippedwith a CoolSNAP-Pro colour digital camera (Olympus).

2.8. Measurement of oxidative stress-induced cell death in zebrafishembryo

Cell death was detected in live embryos using acridine orangestaining, a nucleic acid selective metachromatic dye that interactswith DNA and RNA by intercalation or electrostatic attractions.Acridine orange stains cells with disturbed plasma membrane per-meability so it preferentially stains necrotic or very late apoptotic

Fig. 1. Chemical structures of the five phlorotannins isolated from E. cava.

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Fig. 2. Survival rate after treatment with AAPH or co-treatment with phlorotannins.The embryos were treated with 25 mM AAPH and co-treated with phlorotanninsdieckol (DK), eckstolonol (ES), eckol (EK), triphlorethol A (TA), and phloroglucinol

952 M.-C. Kang et al. / Food Chemistry 138 (2013) 950–955

cells. At 3–4 hpf, the embryos were treated with 50 lM phlorotan-nins and 1 h later, 25 mM AAPH was added to the plate. After treat-ing embryos with 25 mM AAPH for 6 h, the embryo medium waschanged and the embryos developed up to 2 dpf. The embryos weretransferred into 96-well plates and treated with acridine orange(AO) solution (7 lg/ml), and the plates were incubated for 30 minin the dark at 28.5 �C. After incubation, the embryos were rinsedin embryo medium and anaesthetised before visualisation. Individ-ual embryo fluorescence intensity was quantified using a spectro-fluorometer (Perkin–Elmer LS-5B). The images of stained embryoswere observed using a fluorescence microscope, which wasequipped with a CoolSNAP-Pro colour digital camera (Olympus).

2.9. Statistical analysis

All the measurements were made in triplicate and all valueswere represented as mean ± S.E. The results were subjected to ananalysis of variance using the Tukey test to analyse the differences.Values of p < 0.05 were considered significant.

(PG). Experiments were performed in triplicate and the data are expressed asmean ± SE. ⁄⁄p < 0.01.

3. Results

3.1. Toxicity of phlorotannins or AAPH in zebrafish embryo

In order to determine the toxicity of AAPH with or withoutphlorotannins, we monitored the survival rate and growth patternsof zebrafish embryos. The adopted endpoints experiment used toassess the toxicity of the compounds included embryo mortalityand heartbeat disturbances. The survival rate of zebrafish embryostreated with AAPH or co-treated with phlorotannins are shown inFig. 2. A significantly lower survival rate was observed in embryosonly treated with AAPH (around 60% survival rate). However, co-treatment with AAPH and phlorotannins raised the survival rateto 80–90%. The phlorotannins were not associated with mortality

in this experiment. When evaluating the morphological malforma-tions, phlorotannins did not show conspicuous adverse effects,whereas zebrafish embryos exposed to AAPH from 3–4 to 910 hpf(6 h exposure) showed several typical morphological defects. At48 hpf, embryos treated with 25 mM AAPH showed shorter bodylength and, in some cases, incompletely differentiated tail ends,and spinal column curving. Heart curvature was unfolded and a di-lated pericardial sac was observed, suggesting pericardial oedema.Red blood cells accumulated on the side of yolk sac due to circula-tory failure. Yolk consumption was slower than normal, suggestinggrowth retardation. Image analysis data showed that phlorotanninsprotected against morphological changes caused by AAPH. In the

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Fig. 3. Effects of phlorotannins on heartbeat rate. The embryos were treated with25 mM AAPH and co-treated with phlorotannins dieckol (DK), eckstolonol (ES),eckol (EK), triphlorethol A (TA), and phloroglucinol (PG). The heartbeat wasmeasured at 48 hpf. Results are expressed as beats/min. Experiments wereperformed in triplicate and data are expressed as mean ± SE. ⁄p < 0.05.

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heartbeat test, on the other hand, AAPH generated a slight increase,whereas phlorotannins did not generate any heartbeat rate distur-bances as compared with the control (untreated phlorotannins orAAPH, Fig. 3). In simultaneous in vivo toxicity tests, toxicity wasnot detected in the fish treated with phlorotannins, whereas toxicitywas observed in the fish treated with AAPH alone.

3.2. Inhibitory effect of the phlorotannins on AAPH-induced ROSgeneration in zebrafish

The scavenging efficacy of the phlorotannins DK, ES, EK, TA, andPG on ROS production in AAPH-induced zebrafish embryos wasmeasured. Treatment of the embryo with phlorotannins inhibitedROS production (Fig. 4A). Thus, there was a similar ROS level incontrol embryos (without phlorotannins and AAPH) and thosetreated with 50 lM phlorotannins. Fluorescence intensity of the

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Fig. 4. Effect of phlorotannins isolated from E. cava on AAPH-induced ROS level in zebphlorotannins. Photographs of AAPH-induced ROS level in zebrafish embryos (B). ROS leAAPH (positive control), (c) dieckol (DK), (d) eckstolonol (ES), (e) eckol (EK), (f) triphlorethdata are expressed as mean ± SE. ⁄p < 0.05, ⁄⁄p < 0.01.

control embryo was recorded as 1591 whereas the AAPH-treatedembryo showed 3568. However, the addition of phorotanninsto the embryo mixed with AAPH reduced intracellular ROSaccumulation to 1568, 2346, 1703, 1540 and 2262, for DK, ES, EK,TA, and PG, respectively. Fluorescence intensity recovered to a sim-ilar value to that of the control with DK, EK and TA.

Fluorescence photographs of the zebrafish embryo (Fig. 4B)show that the negative control, which contained no phlorotanninor AAPH, generated a clear image, whereas the positive control,treated only with AAPH, generated a fluorescence image, whichsuggests that ROS were formed in the presence of AAPH in the zeb-rafish embryo. However, when the zebrafish embryos were treatedwith phlorotannins prior to AAPH treatment, a dramatic reductionin the amount of ROS was observed. Hence these results indicatethat phlorotannins possess efficient antioxidant properties.

3.3. Lipid peroxidation inhibitory activity

The generation of thiobarbituric acid reactive substances(TBARS) was inhibited in the presence of phlorotannins (Fig. 5A),with similar lipid peroxidation levels in the control (withoutphlorotannins and AAPH) embryos and those treated with 50 lMphlorotannins. Among the phlorotannins, the fluorescence inten-sity was even lower in DK and ES embryos than that of the control.In morphological evaluations, the lipid peroxidation inhibitoryactivity was observed using DPPP fluorescent dye and results areshown in Fig. 5B. The negative control, which contained no phloro-tannin or AAPH, generated a clear image whereas the positive con-trol, only treated with AAPH, generated a fluorescence image,suggesting that lipid peroxidation took place in the presence ofAAPH in the zebrafish embryo. However, when the zebrafish weretreated with phlorotannins prior to AAPH treatment, a dramaticreduction in the amount of lipid peroxidation was observed.

3.4. Protective effects of phlorotannins on AAPH-induced cell death inlive zebrafish

To evaluate whether phlorotannins protect against AAPH treat-ment, cell death induced by AAPH treatment was measured via

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rafish embryos (A). Embryos were treated with 25 mM AAPH and co-treated withvels were measured by image analysis and fluorescence microscopy. (a) Control, (b)ol A (TA), and (g) phloroglucinol (PG). Experiments were performed in triplicate and

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Fig. 5. Effect of phlorotannins isolated from E. cava on AAPH-induced lipid peroxidation level in zebrafish embryos (A). The embryos were treated with 25 mM AAPH and co-treated with phlorotannins. After incubation, the lipid peroxidation was detected by fluorescence spectrophotometer after DPPP staining. Photographs of AAPH-induced lipidperoxidation level in zebrafish embryos (B). The lipid peroxidation levels were measured by image analysis and fluorescence microscopy. (a) Control, (b) AAPH (positivecontrol), (c) dieckol (DK), (d) eckstolonol (ES), (e) eckol (EK), (f) triphlorethol A (TA), and (g) phloroglucinol (PG). Experiments were performed in triplicate and the data areexpressed as mean ± SE. ⁄p < 0.05, ⁄⁄p < 0.01.

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Fig. 6. Protective effect of phlorotannins isolated from E. cava on AAPH-induced cell death in live zebrafish embryos (A). The embryos were treated with 25 mM AAPH and co-treated with phlorotannins. After incubation, the cell death was detected by fluorescence spectrophotometry after acridine orange staining. Photographs of AAPH-induced celldeath in live zebrafish embryos (B). The cell death levels were measured by image analysis and fluorescence microscopy. (a) Control, (b) AAPH (positive control), (c) dieckol(DK), (d) eckstolonol (ES), (e) eckol (EK), (f) triphlorethol A (TA), and (g) phloroglucinol (PG). Experiments were performed in triplicate and the data are expressed asmean ± SE. ⁄p < 0.05, ⁄⁄p < 0.01.

954 M.-C. Kang et al. / Food Chemistry 138 (2013) 950–955

acridine orange as fluorescence intensity in the body of thezebrafish (Fig. 6A). Cell death was reduced by the addition of phloro-tannins to the zebrafish exposed to AAPH. All phlorotannins showedprotective effects against AAPH. Among the phlorotannins, ES andEK showed the highest protective effect against AAPH-induced cell

death in live zebrafish. The microscopic pictures in Fig. 6B show thatthe control zebrafish had intact cells, while AAPH treatment causeda significant increase in the intensity of acridine orange. However,when the fish were treated with phlorotannins prior to AAPH treat-ment, a dramatic decrease in cell death was observed.

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4. Discussion

The physiological benefits of phlorotannins are generallythought to be due to their antioxidant and free radical scavengingproperties, even though phlorotannins display other biologicalactivities (Kang et al., 2003, 2004, 2005, 2010). Although some re-ports suggest that phlorotannins from algae exhibit antioxidant ef-fects against free radicals, there are no reports on the protectiveeffects in zebrafish of phlorotannins (Li et al., 2009). In this study,we investigated the antioxidant effects of phlorotannins, after theadministration of AAPH in zebrafish embryo. 2,7-Dichlorofluores-cein diacetate was used as a probe for ROS measurement. DCFH-DA crosses cell membranes and is enzymatically hydrolysed byintracellular esterase to non-fluorescent DCFH. In the presence ofROS, DCFH is oxidised to highly fluorescent DCF. It is well knownthat AAPH is the principal ROS responsible for the oxidation ofDCFH to DCF (Wang & Joseph, 1999).

In this study, the phlorotannins exhibited ROS scavenging ef-fect, and they were evaluated with regard to their protective ef-fects against AAPH-induced oxidative stress in zebrafishembryos. The results confirm that phlorotannins have an abilityto protect zebrafish embryos from oxidative stress-related cellularinjuries. In a previous study, Kang et al. (2006) reported that somenatural compounds from brown seaweeds have an ability to in-crease levels of catalase, an enzyme located in the peroxisomesof some cells that converts AAPH into molecular oxygen and water.In this study, we showed that phlorotannins have a prominent pro-tective effect on AAPH -induced cell death.

In conclusion, the results obtained in the present study showthat the phlorotannins isolated from E. cava could effectively inhi-bit intracellular ROS formation, lipid peroxidation, and cell deathinduced by AAPH. In addition, we investigated the protective ef-fects of the phlorotannins against AAPH-induced oxidative stressin zebrafish embryos for the first time. Our results demonstratedthat AAPH induces toxicity in zebrafish embryos and phlorotanninscan protect zebrafish embryos against AAPH, by inhibiting intracel-lular ROS formation, lipid peroxidation, and cell death. Thereforezebrafish is a useful in vivo model to elucidate antioxidant effectsof natural compounds.

Acknowledgement

This work was supported by the Korea Science and EngineeringFoundation (KOSEF) grant funded by the Korea government.

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